Pseudomonas monteilii bdlz-3 and application thereof in relieving continuous cropping obstacles of crops

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a Pseudomonas monteilii BDLZ-3 and application thereof in relieving continuous cropping obstacles of crops. The present application discloses a Pseudomonas monteilii BDLZ-3 with a preservation number of CGMCC No.34349. The Pseudomonas monteilii BDLZ-3 disclosed in the present application is first isolated and screened from nature, can efficiently degrade phenolic acid autotoxic substances produced in the process of continuous cropping of ginseng, and the degradation rate of benzoic acid can reach 95.36%, and can also effectively degrade various phenolic acids such as vanillic acid, cinnamic acid, palmitic acid and gallic acid, and has a wide degradation spectrum; and field application of the Pseudomonas monteilii BDLZ-3 disclosed in the present application can effectively reduce the content of benzoic acid in soil, and can also control the occurrence of root diseases of ginseng to a certain extent, with a control effect of 41.23%, and can significantly improve growth indexes such as root length and root thickness and has a yield-increasing effect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Pseudomonas montmorillonii BDLZ-3 and its application in alleviating crop continuous cropping obstacles. Background Technology

[0002] Ginseng ( Panax ginseng Ginseng, a perennial herbaceous plant belonging to the genus *Panax* of the family Araliaceae, is rich in polysaccharides and saponins, possessing various health benefits such as improving eyesight, regulating blood pressure, and enhancing intelligence. It is a valuable traditional Chinese medicine. However, as a perennial herb, ginseng has a long cultivation cycle and requires specific growing environments, which further exacerbates the problem of continuous cropping obstacles. Specifically, this manifests as low seedling survival rates, increased root diseases, and decreased yield and quality, severely hindering the sustainable development of the industry. Therefore, effectively alleviating the continuous cropping obstacles of ginseng has become a crucial issue that urgently needs to be addressed. Summary of the Invention

[0003] The purpose of this invention is to solve the aforementioned problems existing in the prior art and to find an effective solution to alleviate the continuous cropping obstacles of ginseng. To this end, this invention provides a strain of *Pseudomonas montmorillonii* BDLZ-3 and its application in alleviating continuous cropping obstacles.

[0004] This invention provides a strain of Pseudomonas montelukastii ( Pseudomonas monteilii BDLZ-3, with accession number CGMCC No.34349.

[0005] The present invention also provides a method for culturing the aforementioned Pseudomonas montelukastii BDLZ-3, comprising the following steps: inoculating Pseudomonas montelukastii BDLZ-3 into a culture medium for culturing to obtain a culture of Pseudomonas montelukastii BDLZ-3;

[0006] The culture medium comprises the following components in weight percentage: 0.3% to 0.7% yeast extract, 1% to 1.5% soybean peptone, and anhydrous magnesium sulfate.

[0007] The present invention also provides a microbial agent, wherein the effective component of the microbial agent includes Pseudomonas montelukastiensis BDLZ-3 as described in the above technical solution or a culture of Pseudomonas montelukastiensis BDLZ-3 obtained by the culture method described in the above technical solution.

[0008] Preferably, the effective viable count of *Pseudomonas montelukastiensis* BDLZ-3 in the microbial agent is 4 × 10⁻⁶. 7 CFU / mL ~4×10 9 CFU / mL.

[0009] The application further provides the Pseudomonas monteilii BDLZ-3, the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method, or the microbial inoculum in the application of relieving the continuous cropping obstacles of crops.

[0010] Preferably, the relieving the continuous cropping obstacles of crops comprises degrading the autotoxic substances generated by crops in the soil.

[0011] The autotoxic substances comprise at least one of benzoic acid, vanillic acid, cinnamic acid, palmitic acid and gallic acid.

[0012] The application further provides a method for relieving the continuous cropping obstacles of crops, comprising the following steps:

[0013] The soil of the continuous cropping land is treated by the Pseudomonas monteilii BDLZ-3, the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method, or the microbial inoculum.

[0014] Preferably, the treating the soil of the continuous cropping land comprises mixing the Pseudomonas monteilii BDLZ-3, the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method, or the microbial inoculum into the soil.

[0015] The concentration of the strain in the culture or the microbial inoculum is 4×10 7 ~4×10 9 CFU / mL.

[0016] The mixing dose of the culture or the microbial inoculum is 500-1000 mL / m 2 .

[0017] The application further provides the Pseudomonas monteilii BDLZ-3, the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method, the microbial inoculum or the method in any one or more than two functions of ①-④:

[0018] ① reducing the content of benzoic acid;

[0019] ② reducing the occurrence of root diseases of crops;

[0020] ③ improving the growth index of crops;

[0021] ④ improving the yield of crops.

[0022] Preferably, the crop is ginseng.

[0023] Beneficial effects:

[0024] The application provides a Pseudomonas monteilii BDLZ-3, and the preservation number is CGMCC No.34349. The Pseudomonas monteilii BDLZ-3 is first obtained by screening from nature, can degrade phenolic acid autotoxic substances generated by ginseng continuous cropping obstacles, and the degradation rate of benzoic acid can reach 95.36%. Meanwhile, the Pseudomonas monteilii BDLZ-3 can effectively degrade various phenolic acids such as vanillic acid, cinnamic acid, palmitic acid and gallic acid, and has a wide degradation spectrum. Field application of the Pseudomonas monteilii BDLZ-3 can effectively reduce the content of benzoic acid in soil, and can also control the occurrence of ginseng root diseases to some extent, with a control effect of 41.23%, and can significantly improve growth indexes such as root length and root thickness and has a yield-increasing effect.

[0025] Biological preservation information

[0026] The Pseudomonas monteilii BDLZ-3 is classified and named as Pseudomonas monteilii , and was preserved in the China General Microbiological Culture Collection Center (CGMCC) on April 25, 2025, and the address of the CGMCC is No. 1, Beichen West Road, Haidian District, Beijing, and the preservation number is CGMCC No.34349. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0028] Figure 1 It is a statistical diagram of the degradation effect of the strain BDLZ-3 on different phenolic acids;

[0029] Figure 2 It is an observation diagram of the colony morphology, gram staining and cell morphology of BDLZ-3; wherein, A is a colony morphology diagram of the strain BDLZ-3, B is a gram staining result diagram of the strain BDLZ-3, and C is an electron microscope observation diagram of the strain BDLZ-3;

[0030] Figure 3 It is a schematic diagram of a phylogenetic tree of the strain BDLZ-3 constructed based on a 16S rRNA sequence by using a maximum likelihood method;

[0031] Figure 4 It is a result diagram of the influence of initial pH on the growth amount of the strain BDLZ-3 and the degradation effect on benzoic acid;

[0032] Figure 5 It is a result diagram of the influence of substrate concentration on the growth amount of the strain BDLZ-3 and the degradation effect on benzoic acid;

[0033] Figure 6Figure 1 shows the effect of inoculum size on the growth of strain BDLZ-3 and its degradation of benzoic acid.

[0034] Figure 7 Figure showing the effect of culture temperature on the growth of strain BDLZ-3 and its degradation of benzoic acid.

[0035] Figure 8 The figure shows the interaction effect of culture temperature and initial pH on the benzoic acid degradation efficiency of strain BDLZ-3; where a is the contour plot of the interaction effect and b is the response surface plot of the interaction effect.

[0036] Figure 9 The figure shows the interaction effect of inoculum size and initial pH on the benzoic acid degradation efficiency of strain BDLZ-3; where a is the contour plot of the interaction effect and b is the response surface plot of the interaction effect.

[0037] Figure 10 The figure shows the interaction effect of inoculum size and culture temperature on the benzoic acid degradation efficiency of strain BDLZ-3; where a is the contour plot of the interaction effect and b is the response surface plot of the interaction effect.

[0038] Figure 11 The response surface plot and contour plot show the interaction between carbon and nitrogen sources; where a is the contour plot of the interaction and b is the response surface plot of the interaction.

[0039] Figure 12 The response surface plot and contour plot show the interaction between carbon source and inorganic salt; where a is the contour plot of the interaction and b is the response surface plot of the interaction.

[0040] Figure 13 The response surface plot and contour plot show the interaction between nitrogen source and inorganic salt; where a is the contour plot of the interaction and b is the response surface plot of the interaction.

[0041] Figure 14 The graph shows the changes in benzoic acid content in the soil after treatment with strain BDLZ-3. Detailed Implementation

[0042] This invention provides a strain of Pseudomonas montelukastii ( Pseudomonas monteiliiBDLZ-3, and the preservation number is CGMCC No.34349. The Pseudomonas monteilii BDLZ-3 is first isolated and screened from nature, can degrade the phenolic acid autotoxic substance produced by ginseng continuous cropping, and the degradation rate of benzoic acid can reach 95.36%, and can effectively degrade various phenolic acids such as vanillic acid, cinnamic acid, palmitic acid and gallic acid, and has a wide degradation spectrum; and the Pseudomonas monteilii BDLZ-3 can effectively reduce the content of benzoic acid in the soil, and can control the root diseases of ginseng to some extent, and the control effect reaches 41.23%, and can significantly improve the growth indexes such as root length and root thickness and has the yield-increasing effect. The Pseudomonas monteilii BDLZ-3 has white, opaque, round, smooth and neat edge, and the middle of the colony is convex and relatively wet, the cell is short rod-shaped or ellipsoidal, does not produce spores, and is gram-negative. TEM transmission electron microscopy shows that the strain BDLZ-3 is rod-shaped, and the size is (1.5-2.0) μm x (0.5-1.0) μm, and has a flagellum. The Pseudomonas monteilii BDLZ-3 has positive physiological and biochemical test reactions such as starch hydrolysis, indole, fluorescent pigment, hanging drop power, malonic acid and citrate, and has negative test reactions such as oxidase, gelatin liquefaction, methyl red and V-P determination. The 16S rRNA sequence of the Pseudomonas monteilii BDLZ-3 is shown as SEQ ID NO.1:

[0043] Pseudomonas monteilii .

[0044] The application further provides a culture method of the Pseudomonas monteilii BDLZ-3, comprising the following steps: inoculating the Pseudomonas monteilii BDLZ-3 into a culture medium for culture to obtain a culture of the Pseudomonas monteilii BDLZ-3; and the culture medium comprises the following components in percentage by mass: 0.3%-0.7% of yeast powder, 1%-1.5% of soybean peptone and 1%-1.5% of anhydrous magnesium sulfate. As an embodiment, the culture medium comprises the following components in percentage by mass: 0.6% of yeast powder, 1.2% of soybean peptone and 1.4% of anhydrous magnesium sulfate. As another embodiment, the culture medium comprises the following components in percentage by mass: 5.9 g / L of yeast powder, 11.96 g / L of soybean peptone and 13.99 g / L of anhydrous magnesium sulfate. As an embodiment, the culture temperature of the strain BDLZ-3 is 25°C-31°C, the initial pH is 6-8, and the initial inoculation amount is 1%-3%. As another embodiment, the culture temperature of the strain BDLZ-3 is 27.87°C, the initial pH is 7.45, the initial inoculation amount is 2.042%, the rotating speed is 180 r / min, and the bottle filling amount is 100 mL / 250 mL.

[0045] The application further provides a microbial agent, wherein the effective component of the microbial agent comprises the Pseudomonas monteilii BDLZ-3 or the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method according to the above technical solution. As an embodiment, the effective viable cell number of the Pseudomonas monteilii BDLZ-3 in the microbial agent is 4×10 7 CFU / mL-4×10 9 CFU / mL. As another embodiment, the effective viable cell number of the Pseudomonas monteilii BDLZ-3 in the microbial agent is 4×10 8 CFU / mL.

[0046] The application also provides application of the Pseudomonas monteilii BDLZ-3, the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method or the microbial inoculum in relieving crop continuous cropping obstacles. As an implementation form, the crop continuous cropping obstacles relieving method comprises degrading self-toxic substances generated by crops in the soil; the self-toxic substances comprise at least one of benzoic acid, vanillic acid, cinnamic acid, palmitic acid and gallic acid. As an implementation form, the Pseudomonas monteilii BDLZ-3 can degrade 50 μg / mL of benzoic acid by 95.36% in the MSM inorganic salt culture medium taking benzoic acid as the only carbon source, and can effectively degrade various phenolic acids such as vanillic acid, cinnamic acid, palmitic acid and gallic acid, and has a wide degradation spectrum.

[0047] The application further provides a method for relieving crop continuous cropping obstacles, comprising the following steps:

[0048] The soil of a crop continuous cropping field is treated by using the Pseudomonas monteilii BDLZ-3, the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method or the microbial inoculum.

[0049] As an implementation form, the soil of the crop continuous cropping field is treated by mixing the Pseudomonas monteilii BDLZ-3, the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method or the microbial inoculum in the soil; the concentration of the strain in the culture or the microbial inoculum is 4×10 7 ~4×10 9 CFU / mL; and the mixing dose of the culture or the microbial inoculum is 500-1000 mL / m 2 . As another implementation form, the concentration of the strain in the culture or the microbial inoculum is 4×10 8 CFU / mL; and the mixing dose of the culture or the microbial inoculum is 750 mL / m 2 .

[0050] The application further provides application of the Pseudomonas monteilii BDLZ-3, the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method, the microbial inoculum or the method in any one or two or more functions of ①-④:

[0051] ① reducing the content of benzoic acid;

[0052] ② reducing the occurrence of root diseases of crops;

[0053] ③ improving the growth index of crops;

[0054] ④ increasing the yield of crops.

[0055] As an embodiment, the crop according to the present application is ginseng. As an embodiment, the Pseudomonas monteilii BDLZ-3 according to the present application can make the survival rate of ginseng at the harvest period reach 73.08%, which is significantly higher than that of the control group without the treatment of Pseudomonas monteilii BDLZ-3, i.e. 48.08%. As an embodiment, the Pseudomonas monteilii BDLZ-3 according to the present application can alleviate the phenomenon of inhibited growth of ginseng, promote the growth index of ginseng such as root length, root thickness and root fresh weight, and significantly increase the yield of ginseng, with the yield increasing rate reaching 34.10%. As an embodiment, the Pseudomonas monteilii BDLZ-3 according to the present application can effectively control the occurrence of root diseases of ginseng, and the control effect on the root diseases of ginseng (including red skin disease, rust rot disease and root rot disease) reaches 34.42%~41.23%, which has a certain control effect on the root diseases.

[0056] In order to further illustrate the present application, a strain of Pseudomonas monteilii BDLZ-3 and its application in relieving the continuous cropping obstacles of crops according to the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the present application.

[0057] Example 1

[0058] Isolation and screening of strain BDLZ-3

[0059] 1. Isolation and primary screening of bacteria in soil samples

[0060] Through the enrichment domestication and re-isolation method, 213 strains of bacteria were obtained from 60 soil samples collected from the ginseng continuous cropping land. The 213 strains of bacteria were subjected to primary screening by determining the degradation ability of the bacteria on benzoic acid by using the ultraviolet spectrophotometer method, and 6 strains of bacteria with the benzoic acid degradation rate of more than 80% were obtained.

[0061] 2. Secondary screening of bacteria in soil samples

[0062] The 6 strains of bacteria obtained by primary screening were subjected to secondary screening by using the high performance liquid chromatography method, and the degradation effect and degradation spectrum of the 6 strains of bacteria on benzoic acid and other soil phenolic acid autotoxic substances (vanillic acid, cinnamic acid, palmitic acid and gallic acid) were further determined. The chromatographic analysis was performed by using an Aichrom Bond-AQ C18 chromatographic column (4.6 mm x 250 mm, 5 µm) with acetonitrile-methanoic acid aqueous solution (volume ratio 80:20) as the mobile phase, wherein the methanoic acid aqueous solution was 0.1% (v / v) methanoic acid aqueous solution, the flow rate was 0.8 mL / min, the ultraviolet detection wavelength was 230 nm, the column temperature was 30℃, the injection amount was 10 µL, and the isocratic elution was performed.

[0063] The test strain was placed in LB liquid medium for culture, and 2% (volume fraction) bacterial suspension was taken OD 600 =1.0) was inoculated in MSM-BA medium containing 50 mg / L, and MSM-BA culture solution without adding bacteria was used as a control. After 3 days, 10 mL was taken each time, extracted with an equal volume of ethyl acetate for 3 times, evaporated to dryness with a rotary evaporator, and then 10 mL of methanol was added to dissolve the solid material, and the solution was filtered with a 0.22 µm filter. The degradation rate (D) D ) was calculated according to formula I.

[0064] , formula I;

[0065] In the formula, Co is the initial concentration of the sample (mg / L), Ct is the detected concentration of the sample (mg / L).

[0066] After re-screening, only one strain BDLZ-3 from the 6 primary screening strains had certain degradation activity on vanillic acid, cinnamic acid, palmitic acid and gallic acid. The degradation rate of strain BDLZ-3 on 5 kinds of phenolic acids such as benzoic acid, vanillic acid, cinnamic acid, palmitic acid and gallic acid is shown in Figure 1 and Table 1. It can be seen that in addition to having high degradation activity on benzoic acid, the strain also has obvious degradation effect on vanillic acid and gallic acid, and the degradation rates are 90.42% and 61.75%, respectively.

[0067] Table 1 Degradation effect of strain BDLZ-3 on phenolic acids

[0068]

[0069] Example 2

[0070] Identification of strain BDLZ-3

[0071] 1. Morphological identification

[0072] The colony morphology of the degradation strain BDLZ-3 on NA plate was observed, and the strain was subjected to gram staining and transmission electron microscope observation. The colony morphology of strain BDLZ-3 is shown in A of Figure 2 , the gram staining result of strain BDLZ-3 is shown in B of Figure 2 , and the electron microscope observation result of strain BDLZ-3 is shown in C of Figure 2 .

[0073] It can be seen that the strain BDLZ-3 is white, non-transparent, round, smooth and neat edge, with a convex in the middle of the colony, relatively wet, and the cell is short rod or ellipsoid, and does not produce spores, and is gram-negative on NA medium. TEM transmission electron microscopy shows that the strain BDLZ-3 is rod-shaped, with a size of (1.5-2.0) μm x (0.5-1.0) μm, and has a flagellum.

[0074] 2. Physiological and biochemical identification

[0075] The morphological characteristics and physiological and biochemical properties of the strain were determined according to the Manual of Bacterial Identification and the Systematic Identification Manual of Common Bacteria. The physiological and biochemical determination results of the strain BDLZ-3 are shown in Table 2, wherein "+" represents a positive reaction; and "-" represents a negative reaction. The physiological and biochemical determination results show that the strain BDLZ-3 is positive in starch hydrolysis, indole, fluorescent pigment, hanging drop power, malonic acid, and citrate test (Table 2), and in combination with the morphological characteristics and physiological and biochemical property test results, and in reference to the Manual of Bacterial Identification, the strain BDLZ-3 is preliminarily identified as Pseudomonas sp..

[0076] Table 2 Physiological and biochemical determination results of the strain BDLZ-3

[0077]

[0078] 3. Molecular biology identification

[0079] The bacterial kit for molecular biology identification (D1600-100, Beijing Solabio Science and Technology Co., Ltd.) was used to extract the bacterial DNA for molecular biology identification of the degrading bacteria, and the nucleotide sequences of the PCR reaction primers (27F / 1492R) are shown in SEQ ID NO. 2 and SEQ ID NO. 3;

[0080] SEQ ID NO. 2: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0081] SEQ ID NO. 3: 5'-TACGGTTACCTTGTTACGACTT-3'.

[0082] The PCR product was sent to Shengong for sequencing, sequence analysis and homology comparison were performed by using MEGA 11.0 software, and Bootstrap method (1000 times of repetition) was used for testing, and Escherichia coli strain U 5 / 41 NR 024570.1 as an outgroup, and the maximum likelihood method (Maximum Likelihood) was used for constructing the phylogenetic tree, and the identification phylogenetic tree of the strain BDLZ-3 is shown in Figure 3As shown, the measured 16S rRNA sequence was submitted to GenBank.

[0083] The 16S rRNA sequence of strain BDLZ-3 is shown in SEQ ID NO.1.

[0084] Using DNA from the degrading strain BDLZ-3 as a template, PCR amplification was performed using universal primers for the bacterial 16S rRNA gene. The sequence was compared using EzBioCloud and NCBI, and the results showed that the degrading strain BDLZ-3 was similar to *Pseudomonas montelukastii* (…). Pseudomonas monteilii DB13 MN889014.1) belong to the same branch. Finally, based on morphological observation, physiological and biochemical index determination, and 16S rRNA sequence analysis, strain BDLZ-3 was identified as a strain of Pseudomonas montelukastii (…). Pseudomonas monteilii It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 25, 2025, with accession number CGMCC NO.34349.

[0085] Example 3

[0086] Optimization of benzoic acid degradation conditions and culture medium formulation for strain BDLZ-3

[0087] The tested strain BDLZ-3 was inoculated into LB liquid medium and cultured with shaking for 12 h (30℃, 180 r·min⁻¹) to obtain a bacterial suspension. The bacterial suspension was then inoculated into an inorganic salt medium containing 50 mg·L⁻¹ benzoic acid for single-factor experiments. The pH (5.0, 6.0, 7.0, 8.0, 9.0), temperature (25℃, 28℃, 31℃, 34℃, 37℃), inoculum size (1%, 2%, 3%, 4%, 5%), and substrate concentration (10 mg·L⁻¹) were measured. -1 30 mg·L -1 50 mg·L -1 70 mg·L -1 90 mg·L -1 The effects of each single factor on the degradation efficiency of the strain were investigated. After 3 days of cultivation, the growth rate and benzoic acid degradation efficiency of the strain were measured to clarify the influence of each factor on the degradation efficiency. The effects of initial pH, substrate concentration, culture temperature, and inoculum size on the growth and benzoic acid degradation ability of strain BDLZ-3 were obtained as follows: Figures 4-7 As shown, where, Figure 4 The figure shows the effect of initial pH on the growth of strain BDLZ-3 and its degradation effect on benzoic acid. Figure 5 The figure shows the effect of substrate concentration on the growth of strain BDLZ-3 and its degradation effect on benzoic acid. Figure 6The results of the influence of inoculum amount on the growth amount of strain BDLZ-3 and the degradation effect of benzoic acid are shown in the figure; Figure 7 The results of the influence of culture temperature on the growth amount of strain BDLZ-3 and the degradation effect of benzoic acid are shown in the figure. Figures 4-7 The results shown in the figure show that when the pH is 6-8, the substrate concentration is 50-90 mg·L -1 , the temperature is 28℃, and the inoculum amount is 2%, the degradation effect of strain BDLZ-3 on benzoic acid is the best, the degradation rate is greater than 80.00%, and the growth of the bacterial cells is good.

[0088] 1. Degradation condition optimization test

[0089] On the basis of the single-factor test, the culture temperature (25℃, 28℃, 31℃), the initial pH (6, 7, 8), and the initial inoculum amount (1%, 2%, 3%) were determined as the three main factors affecting the degradation rate of benzoic acid. Box-Behnken design was used to optimize the three key factors of pH, temperature, and inoculum amount. A 3-factor 3-level test was designed, and the levels of each factor were coded according to the requirements of Box-Behnken design. The coded values of pH (6, 7, 8), temperature (25℃, 28℃, 31℃), and inoculum amount (1%, 2%, 3%) were -1, 0, and 1, respectively. The specific test design and grouping are shown in Table 3.

[0090] Strain BDLZ-3 was added to MSM inorganic salt medium with benzoic acid as the sole carbon source (benzoic acid content 50 µg / mL), and the same MSM medium without strain BDLZ-3 was used as the control. After 3 days of culture, the degradation of benzoic acid was detected to obtain the degradation rate of benzoic acid, as shown in Table 3.

[0091] Table 3 Box-Benhnken test design based on 3 factors and 3 levels

[0092]

[0093] The quadratic polynomial regression model established by fitting is shown in formula II:

[0094] Y BDLZ-3 =95.3-0.8775* A -0.1675* B +0.0925* C -0.0250* AB +0.2650* AC -0.045* BC -3.48* A 2 -2.06* B 2 -2.545*C 2 , formula II.

[0095] Wherein, A, B, C in formula II respectively represent the coding value of factors pH, temperature (℃) and inoculation amount (%), Y is the degradation rate of benzoic acid. The model R 2 =99.54%, P =(0.0001)<0.05 extremely significant, the loss of fitting term = 0.0787 > 0.05 not significant, which shows that the model is meaningful and the fitting degree of test data is good, the probability that the test data does not conform to the model is very small, and the model can be used for theoretical prediction. The interactive influence results of inoculation amount, culture temperature and initial pH on the degradation efficiency of benzoic acid by strain BDLZ-3 are obtained by software analysis as shown in Figures 8-10 Figure 8 is the interactive influence results graph of culture temperature and initial pH on the degradation efficiency of benzoic acid by strain BDLZ-3, including the interactive influence contour graph as shown in a and the response surface graph as shown in b; Figure 9 is the interactive influence results graph of inoculation amount and initial pH on the degradation efficiency of benzoic acid by strain BDLZ-3, including the interactive influence contour graph as shown in a and the response surface graph as shown in b; Figure 10 is the interactive influence results graph of inoculation amount and culture temperature on the degradation efficiency of benzoic acid by strain BDLZ-3, including the interactive influence contour graph as shown in a and the response surface graph as shown in b. Thus, the optimal value points of the three main factors are respectively pH 7.454, temperature 27.866 ℃ and inoculation amount 2.042%, and under this condition, the predicted degradation rate is 94.193%, the average value of 3 actual test results is 95.36%, which is higher than the predicted value, and the fitting degree of test value and predicted value is relatively high, which shows that the established model is effective (Table 4).

[0096] Table 4: Response surface test results

[0097]

[0098] 2. Medium formula optimization test

[0099] The test strain BDLZ-3 was inoculated in LB liquid medium and oscillated for 12 h (30 ℃, 180 r·min-1) to obtain bacterial suspension, and the bacterial suspension was inoculated in LB liquid medium containing 50 mg·L -1 ​The effects of carbon source (0.1%, 0.3%, 0.5%, 0.7%, 0.9%), nitrogen source (0.5%, 0.75%, 1%, 1.25%, 1.5%) and inorganic salt (0.5%, 0.75%, 1%, 1.25%, 1.5%) on the growth of the strain were determined in the inorganic salt medium of benzoic acid, and the effects of each single factor on the production of the strain were finally determined after 3 days of culture to determine the optimal medium formula as yeast powder, peptone and anhydrous magnesium sulfate. The addition amounts of yeast powder, soybean peptone and anhydrous magnesium sulfate were 0.3%, 0.5%, 0.7%, 1%, 1.25%, 1.5% and 1%, 1.25%, 1.5%, respectively, and the Box-Behnken design was used to optimize the coding levels of yeast powder, soybean peptone and anhydrous magnesium sulfate. The coding levels of the test factors and the corresponding actual concentrations are shown in Table 5, and the test design is shown in Table 6.

[0100] Table 5 Box-Behnken design test factors and coding levels

[0101]

[0102] The strain BDLZ-3 was added to the MSM-BA medium, and the same MSM medium without the addition of the strain BDLZ-3 was used as a control. After 1 day of culture, the OD600 of the strain BDLZ-3 was detected (Table 6).

[0103] Table 6 Box-Benhnken test design

[0104]

[0105] The response surface method was used to optimize the three factors and three levels of carbon source, nitrogen source and inorganic salt, and the obtained quadratic polynomial regression equation was:

[0106] Y BDLZ-3 =1.95+0.0574* A -0.0235* B +0.0356* C +0.0087* AB +0.0564* AC +0.0010* BC -0.0997* A 2 -0.0438* B 2 -0.0424* C 2 , formula III.

[0107] Wherein, A, B, C in formula III respectively represent the coding value of the addition amount of yeast powder, soybean peptone and anhydrous magnesium sulfate, and Y is the response value of the growth amount of strain BDLZ-3, that is, OD600.

[0108] Table 7 Variance analysis results of response surface design

[0109]

[0110] The variance analysis results of the response surface design are shown in Table 7. As shown in Table 7, the model is significant, and the fitting degree of the experimental data is good, and the probability that the experimental data does not conform to the model is very small, so the model can be used for theoretical prediction. R 2 =95.85%; P =0.0005<0.01 extremely significant, and the lack of fit term 0.2529>0.05 is not significant, indicating that the model is meaningful and the fitting degree of the experimental data is good, and the probability that the experimental data does not conform to the model is very small, so the model can be used for theoretical prediction. Using software analysis, the interactive influence results of carbon source, nitrogen source and inorganic salt are shown in Table 8, wherein Figures 11-13 Figure 11 is the response surface graph (b) and the contour graph (a) of the interactive influence of carbon source and nitrogen source; Figure 12 is the response surface graph (b) and the contour graph (a) of the interactive influence of carbon source and inorganic salt; Figure 13 is the response surface graph (b) and the contour graph (a) of the interactive influence of nitrogen source and inorganic salt. Thus, the optimal value points of the three main factors are respectively yeast powder 5.9 g / L, soybean peptone 11.96 g / L, and magnesium sulfate 13.99 g / L. Under these conditions, the predicted OD 600 is 1.953, and the average value of the three actual tests OD 600 is 2.099, which is higher than the predicted value, and the fitting degree of the experimental value and the predicted value is relatively high, indicating that the established model is effective.

[0111] In summary, the optimal fermentation medium formula of strain BDLZ-3 is determined as follows: yeast powder (5.90 g / L), soybean peptone (11.96 g / L), and anhydrous magnesium sulfate (13.99 g / L), and under the culture conditions of pH 7.45, temperature 27.87°C, inoculum size 2.04%, rotation speed 180 r / min, and bottle filling amount 100 mL / 250 mL, the degradation rate of 50 µg / mL benzoic acid is 95.36%, and the biomass of the bacteria after optimization reaches 6.5×10 10 cfu / mL, which is increased by 13.0 times.

[0112] Example 4

[0113] Field application test of benzoic acid degrading bacteria BDLZ-3

[0114] ​The degradation capacity of the bacterium BDLZ-3 on benzoic acid in ginseng-continuously cropped soil was evaluated under field conditions. The experiment was conducted at the ginseng experimental base of Jilin Agricultural University in Jingyue District, Changchun City. The bacterial cell count was 4 × 10⁻⁶. 9 4×10 8 and 4×10 7 CFU·mL -1 The fermentation broth of strain BDLZ-3 was fermented at 750 mL / m 2 The dosage was evenly mixed into the soil. Five treatments were included: a 2% biochar treatment and a control with an equal volume of fermentation medium. There were three replicates. The plot area was 2m². 2 Healthy ginseng plants with uniform growth were selected for transplanting. Samples were taken 120 days after treatment, and the change in benzoic acid content in the soil was detected using the Martens method. At harvest, agronomic traits and disease incidence of the ginseng were investigated and measured. The grading method for root diseases is shown in Table 8, and the calculation of the disease index and control efficacy is shown in Equations IV and V.

[0115] Table 8 Grading Standards for Ginseng Root Diseases

[0116]

[0117] Disease index = Σ (number of disease roots at each level × representative value at each level) / (total number of disease roots surveyed × highest representative value) × 100, Equation IV;

[0118] Prevention and control effect (%) = (disease index of control area - disease index of treatment area) / disease index of control area × 100, formula V.

[0119] 1. Detection of benzoic acid content in soil

[0120] The changes in benzoic acid content in soil were detected using the Martens method, and the results of changes in benzoic acid content in soil after different treatments were obtained as follows: Figure 14 As shown in the figure. After measuring the benzoic acid content in the soil, it was found that the benzoic acid content in the soil treated with the control culture medium but not inoculated with strain BDLZ-3 remained almost unchanged at 7.30 mg·kg⁻¹. -1 The concentration of benzoic acid decreased to some extent in the uninoculated biochar treatment, with a benzoic acid content of 5.35 mg·kg⁻¹. -1 The degradation rate was 17.43%, but the effect was not significant. In contrast, the benzoic acid in the soil of the three treatments inoculated with BDLZ-3 showed varying degrees of degradation, with levels of 3.63 mg / kg. -1 3.01 mg·kg -1 3.90 mg·kg -1 The degradation rates of benzoic acid were 44.17%, 57.52%, and 49.18%, respectively, with the highest rate being 4.0 × 10⁻⁶. 8 CFU·mL -1BDLZ-3 had the best degradation effect.

[0121] 2. The effect of strain BDLZ-3 on the growth of ginseng

[0122] The statistical results of the agronomic traits of ginseng treated with strain BDLZ-3 are shown in Table 9. It can be seen that the addition of different concentrations of strain BDLZ-3 fermentation broth can promote the growth of ginseng. Among them, the effect of BDLZ-3 with a concentration of 4×10 8 CFU / mL is the best, and there is a significant difference at the 5% level compared with the control treatment. At the same time, the survival rate of ginseng at the harvest period is 65.38%~73.08%, which is significantly higher than that of each control treatment, and there is a significant difference at the 5% level. It shows that after treating BDLZ-3 in the soil containing benzoic acid, it can alleviate the inhibition of ginseng growth, and each growth index is improved.

[0123] Table 9 Agronomic traits of ginseng treated with strain BDLZ-3

[0124]

[0125] Note: CK1 and CK2 represent 2% biochar and medium treatment, respectively, BBA1, BBA2 and BBA3 represent inoculation of BDLZ-3 4×10 9 , 4×10 8 , 4×10 8 CFU·mL -1 of liquid, all taking CK2 as the control to calculate the yield increase. The same column data after different lowercase letters represent the application of Duncan new multiple range test at P <0.05 level difference is significant, (⁻x±s, n=3).

[0126] 3. The effect of strain BDLZ-3 on the occurrence of root diseases of ginseng

[0127] According to the classification method of Table 8, formula IV and formula V, the occurrence of ginseng diseases treated with strain BDLZ-3 is shown in Table 10. It can be seen that the occurrence of root diseases of ginseng (including red skin disease, rust rot disease, root rot disease) is different. Among them, the medium control has the most serious disease, and the root disease is relieved after inoculation of strain BDLZ-3. Compared with the medium control, the control effect is 34.42%, 41.23% and 35.88% respectively. Biochar can reduce the content of benzoic acid in soil, but it cannot effectively prevent the occurrence of root diseases.

[0128] Table 10 Occurrence of ginseng diseases treated with strain BDLZ-3

[0129]

[0130] Note: CK1, CK2 represent 2% biochar, medium treatment respectively, BBA1, BBA2, BBA3 represent inoculation of BDLZ-3 4×10 9 , 4×10 8 , 4×10 7 CFU·mL -1 strain liquid 750 mL treatment, all with CK2 as control to calculate the control effect; different lowercase letters represent the application of Duncan new multiple range test in P <0.05 level significant difference (x±s, n=3).

[0131] As can be seen from the above, the strain BDLZ-3 fermentation liquid at 4.0×10 8 CFU / mL of the highest degradation rate of benzoic acid reached 57.52%, significantly higher than the biochar control; and the field application of degradation strain BDLZ-3 can control the occurrence of root disease to a certain extent, the control effect of root disease reaches 34.42%~41.23%, can significantly improve the growth index of ginseng root length, root thickness, etc., has a certain yield-increasing effect.

[0132] Although the above embodiments make a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and people can also obtain other embodiments according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. A strain of Pseudomonas montelukastii ( Pseudomonas monteilii BDLZ-3, characterized in that, The preservation number is CGMCC No. 34349.

2. The culture method of Pseudomonas monteilii BDLZ-3 according to claim 1, characterized by, The method comprises the following steps: The Pseudomonas monteilii BDLZ-3 is inoculated into a culture medium for culture to obtain a culture of the Pseudomonas monteilii BDLZ-3; The culture medium comprises the following components in mass percentage: 0.3%-0.7% of yeast powder, 1%-1.5% of soybean peptone, and 1%-1.5% of anhydrous magnesium sulfate.

3. A microbial inoculant, characterized in that, The effective component of the microbial agent comprises the Pseudomonas monteilii BDLZ-3 of claim 1 or the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method of claim 2.

4. The microbial inoculant of claim 3, wherein, The effective viable cell number of Pseudomonas monteilii BDLZ-3 in the microbial inoculant is 4×10 7 CFU / mL~4×10 9 CFU / mL.

5. The Pseudomonas monteilii BDLZ-3 of claim 1, the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method of claim 2, or the microbial agent of claim 3 or 4 is applied to alleviate the continuous cropping obstacle of ginseng.

6. Use according to claim 5, characterized in that, The alleviation of the continuous cropping obstacle of ginseng comprises degradation of self-toxic substances generated by ginseng in soil; The self-toxic substances are at least one of benzoic acid, vanillic acid, cinnamic acid, palmitic acid, and gallic acid.

7. A method for alleviating ginseng continuous cropping obstacles, characterized in that, The method comprises the following steps: The soil of the continuous cropping land of ginseng is treated by using the Pseudomonas monteilii BDLZ-3 of claim 1, the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method of claim 2, or the microbial agent of claim 3 or 4.

8. The method of claim 7, wherein, The treatment of the soil of the continuous cropping land of ginseng comprises mixing the Pseudomonas monteilii BDLZ-3 of claim 1, the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method of claim 2, or the microbial agent of claim 3 or 4 into the soil; The concentration of the strain in the culture or microbial inoculant is 4 x 10 7 ~4 x 10 9 CFU / mL. The culture or microbial inoculant is mixed at a dosage of 500-1000 mL / m 2 .

9. The Pseudomonas monteilii BDLZ-3 of claim 1, the culture of the Pseudomonas monteilii BDLZ-3 obtained by the culture method of claim 2, the microbial agent of claim 3 or 4, or the method of claim 7 or 8 is applied to any one or more than two functions of ①-③: ①reducing root diseases of ginseng; ②improving growth indexes of ginseng; ③increasing yield of ginseng; The root diseases of ginseng are red skin disease, rust rot disease, and root rot disease; The growth indexes of ginseng are survival rate, average root length, average root thickness, and root fresh weight.

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